The document describes planar chromatography techniques, specifically thin layer chromatography (TLC). It explains that TLC separates mixtures by using a thin stationary phase like silica gel coated on a plate and a mobile phase liquid solvent. The steps of TLC are described as sample application, development where separation occurs, visualization under UV light, and interpretation by calculating Rf values. Applications for separating lipids, carbohydrates and other compounds are outlined.

1. PLANAR CHROMATOGRAPHY Ferosekhan . S FNB-41

12. Thin-Layer Chromatography: A Two-Component Mixture More polar! Less polar! mixture s o l v e n t f r o n t c o m p o n e n t B c o m p o n e n t A o r i g i n s o l v e n t f r o n t c o m p o n e n t B c o m p o n e n t A o r i g i n Increasing Development Time s o l v e n t f r o n t o r i g i n

13. Thin Layer Chromatography TLC plate silica gel - silicon dioxide (SiO 2 ) x (a common, inexpensive stationary phase) bulk (SiO 2 ) x These exposed OH units give silica gel a relatively polar surface. surface

14. Four Stages in TLC 1. Sample Application - Capillary used to spot solution of each sample. 2. Development - This is when the separation actually occurs. 3. Visualization - viewed under UV light. 4. Interpretation of Result - Comparison of retention factors.

15. 1. Sample Application (spotting) TLC plate B. Dissolve solid sample in MeOH C. Use TLC capillary to transfer and spot dissolved sample Sample A B C Ref. Ref. Ref. “ finishing line”  1 cm. A. Draw “guide lines” lightly with pencil “ starting line”  1 cm.

16. 2. Development of TLC Plate TLC Developing Chamber (just a glass jar with solvent in it!) A. Place spotted TLC plate in developing chamber B. Developing solution is drawn up the plate by capillary action C. Remove TLC plate when solvent reaches top line Developing solution (mobile phase) } {keep capped} NOTE: During this ~20 min. developing stage, compounds in the original spots are being pulled through the silica gel. TLC plate

17. 3. Visualization of TLC Results A. Allow solvent to evaporate from surface of TLC plate. C. Mark spots with a pencil while viewing under UV. UV B. View results under UV light . look for grayish spots on the fluorescent green background

18. 4. Interpretation of TLC Results A. Determine retention factors (R f ) for each spot detected. B. Use R f ’s of reference spots to identify the other components. How do you interpret any other spots? 4 1 3 2 ? ? distance spot has moved distance solvent has moved _______________________ R f = = X Y Y - - - - - - - - - - - - - - - - - - - - - - - X 3 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - X 1 X 2

20. Separation of Total Lipid into different Classes Mobile Phase: hexane: diethyl ether: formic acid (80:20:2) Cholesteryl esters TAG Free fatty acids Cholesterol 1,3-DAG 1,2-DAG Monoacyl glycerols Phospholipids

21. Separation of Triacylglycerols Mobile Phase: Pet ether: diethyl ether: acetic acid (90:9:1) Tristearin 2-oleodistearin 1-stereodiolein Triolein Trolinolein With HUFA

24. Paper Chromatography